Human papillomaviruses (HPVs) cause a variety of human diseases including genital warts and cervical carcinomas. Replication of HPV genomes requires virus-encoded E1 and E2 proteins, whereas transcription of HPV genes is mainly regulated by E2 and cellular transcription factors. To investigate the mechanisms of HPV transcription, we have developed two cell-free transcription systems reconstituted either with individually purified general transcription factors and cofactors or with a preassembled RNA polymerase II holoenzyme and TFIID (or TBP). Interestingly, both activation and repression of the HPV E6 promoter can be recapitulated in vitro by differential amounts of E2 proteins as observed previously in transfected cells. This provides us with a unique opportunity to dissect the mechanisms of transcriptional regulation mediated by E2 and to define the role o cellular proteins in HPV transcription. The objectives in this proposal are: 1) To define the role of cellular proteins in mediating the functions of HPV E2 proteins. The observation that E2-mediated activation and repression can both occur in our highly purified transcription system reconstituted with only recombinant general transcription factors and cofactors (TBP, TFIIB, TFIIE, TFIIF, and PC4) and epitope-tagged multiprotein complexes (TFIID, TFIIH, and RNA polymerase II) suggests that components of the general transcription machinery are the targets of E2-mediated regulation. To define the steps of transcription complex assembly regulated by E2 proteins, we will perform template challenge and order-of-addition experiments as well as functional recruitment assays with our cell-free transcription systems using both synthetic DNA templates containing multimerized E2-binding sites and natural HPV templates containing the E6 promoter linked to the upstream regulatory region (URR). 2) To dissect the mechanism of HPV chromatin transcription mediated by various viral and cellular proteins. Since many gene-specific transcription factors and cofactors only work in the context of chromatin, we will develop an in vitro chromatin transcription system to study the mechanisms by which viral E2 and cellular enhancer-binding factors transcribe HPV chromatin. The Drosophila S190 chromatin assembly extract and purified histones will be used to assemble HPV-11 DNA templates, which will then be characterized by micrococcal nuclease digestion and Southern blotting with promoter-proximal and -distal DNA probes, and used for in vitro transcription to define the role of E2 and enhancer-binding factors in HPV chromatin transcription. These studies will uncover the role of cellular proteins in mediating E2 functions and further shed light on the molecular mechanism of HPV transcription.